Effect Of Hyperlipidemia On Homing Efficiency Of Systemically Transplanted Bone Marrow Stromal Cells And Bone Regeneration In Apoe Deflcient MICE

Introduction:Bone defects in the oral and maxillofacial region usually interfere with normal masticatory function and often have devastating esthetic, emotional and social impact on patients. Physiological and functional reconstruction of the damaged tissues is highly expected, which requires not only the differentiation of local reparative cells but also the recruitment of mesenchymal stem cells (MSCs) from bone marrow and peripheral circulation. In response to the injury stimuli, a series of cellular and molecular events are activated, various cytokines and growth factors are released by platelets and inflammatory cells, and eventually the MSCs are induced to migrate into the wound site. Recruited MSCs then proliferate and differentiate into osteoblasts and chondrocytes which actively participate in bone regeneration.Bone marrow stromal cells (BMSCs) are a type of pluripotent mesenchymal stem cells with the capacity for multipotent differentiation into tissues of mesodermal origin such as fat, bone, cartilage, ligament and more. As the progenitor cells for osteoblasts, BMSCs have always played a substantial role in reconstitution of bone tissue. After systemic transplantation into irradiated mice with mandibular bone defects, BMSCs can be detected in the wound sites, undergo osteogenic differentiation and significantly promote local bone regeneration. Although the mechanisms responsible for the recruitment of BMSCs have not yet been fully revealed, accumulating lines of evidence suggest that cytokines and chemokines probably play critical roles in the mobilization of BMSCs and their subsequent homing to the injured tissues. However, currently it is still unclear whether systemic health conditions directly affect the homing efficiency of BMSCs.An association between hyperlipidemia and osteoporosis has been suggested by a variety of studies. Epidemiologic evidence indicates the elevated serum lipid level as a risk factor of osteoporosis. Furthermore, diet-induced hyperlipidemia leads to significantly reduction in bone mineral density (BMD) and bone mineral content (BMC) in animal models, and lipid-lowering treatments increase BMD and lower fracture risk in humans. Hyperlipidemia impaired bone regeneration through actions of oxidized lipids. When treated with the oxidation products of low density lipoprotein (LDL) in vitro, BMSCs isolated from hyperlipidemic individuals underwent an adipogenic differentiation instead of osteogenic differentiation. Unfortunately, little is known whether hyperlipidemia disturbs the recruitment of BMSCs to the bone defect sites from peripheral circulation and further inhibits bone regeneration. In this study, hyperlipidemia was established using an ApoE deficient (ApoE-/-) mouse model. Mandibular bone defects were then created in these mice and BMSCs labeled with the green fluorescent protein (GFP) were transplanted via the tail vein. The homing efficiency of transplanted BMSCs was evaluated under fluorescence microscopy and the new bone formation was determined using histomorphometric analysis.-Materials and methods:1. BMSCs were isolated from C57BL/6J mice by bone marrow adherent method. BMSCs at passage4were incubated with antibodies against mouse CD34, CD44, CD45, CD29for flow cytometry analysis. CFU-F assay was performed to assess the colony-forming efficiency of BMSCs. To assess the multipotent differentiation potential, BMSCs were incubated in a-MEM containing5%fetal calf serum (FCS),10mM β-glycerophosphate,50mg/L ascorbate-2-phosphate and0.1μM dexamethasone for osteogenic differentiation induction;10%FCS,1μM dexamethasone,200μM indomethacin,10μM insulin and0.5mM isobutyl-methylxanthine (IBMX) for adipogenic differentiation induction and10%FCS,50nM ascorbate-2-phosphate,0.1μM dexamethasone for chondrogenic differentiation induction. Alizarin red staining, oil red O staining and toluidine blue staining were performed to examine the osteogenic, adipogenic and chondrogenic differentiation respectively. To track directly the migration and differentiation of BMSCs in vivo, the lentiviral vector with enhanced green fluorescent protein (GFP) was used to label BMSCs. Transfected cells were purified with G418(100ng/ml).2. Eight-week-old male ApoE-/-mice on C57BL/6J background and C57BL/6J mice were fed on a high-fat/high-cholesterol/cholate diet (15%/1.25%/0.5%, respectively). Body weight of these animals was recorded every week. Fasting blood samples were taken from the angular vein of8-wk-old and12-wk-old animals to determine serum lipid levels including LDL, high density lipoprotein (HDL), triglyceride (TG) and total cholesterol (TC). Bone defects were created in the mandibles and BMSCs labeled with GFP were then injected via the tail vein. Mice in control groups were injected with a-MEM of the same volume. The animals were sacrificed at weeks1,2, and4after surgery. The fate of the transplanted BMSCs was monitored with a fluorescence microscope and immunohistochemical analysis. After hematoxylin and eosin (HE) staining and Masson’s Trichrome (MT) staining, histomorphometric analysis was performed to evaluate bone regeneration.Results:1. Primary BMSCs with stable morphology was obtained and continuously subcultured in vitro. Flow cytometry analysis showed that the4th generation BMSCs expressed CD44+(96.48%), CD29+(97.98%), CD31-and CD45-, which are markers for BMSCs, indicating the cultured BMSCs were of high purity. CFU-F assay showed BMSCs had strong colony forming ability. After osteogenic, adipogenic and chondrogenic differentiation induction, mineralized nodules, accumulated lipids and the positive staining density were detected with alizarin red staining, red O staining and toluidine blue staining respectively, which indicated that BMSCs owned the multipotent differentiation potential. GFP could be detected48h post-transfection in BMSCs and expressed stably as the cell passage increased.2. After fed with the HFD for4weeks, serum lipid levels were dramatically higher in ApoE-/-mice than in C57BL/6J mice. Briefly, ApoE-/-mice showed an8.1-fold increase in TC levels and a14.4-fold increase in LDL levels when compared with the control mice, indicating a diet-induced hyperlipidemia in ApoE-/-mice was successfully established. Body weight of both ApoE-/-and C57BL/6J mice increased continuously. No significant difference in body weight was detected between the two mouse strains all through the experimental period. Fluorescence microscope observation and immunohistochemical analysis showed the number of GFP-positive BMSCs detected in the bone defects reached its peak at1week after surgery and was decreased thereafter in both groups transplanted with BMSCs. However, at all time points, less GFP+cells were detected in the ApoE-/-mice than in the corresponding control mice. In the2nd and4th week, GFP+osteoblasts were seen in the surface and inside of newly formed bone which further indicated transplanted BMSCs can home to the mandibular defect and participate in the bone regeneration. At one week post-operation, bone defects in all of the four groups were filled with abundant connective tissue containing large numbers of fibroblasts and a small amount of inflammatory cells. At two weeks after surgery, collagen fiber build-up was evident and some small islands of osteoid were also detected in the center of the bone defects. Newly formed woven bone were observed on the margins and extended towards the center of the defect. By the fourth week, all groups showed advanced bone formation and calcification. Observation of the MT-stained sections showed large area of dark blue color in all groups and reddish matured bone was identified in group C. Histomorphometric analysis showed both at2weeks and4weeks the area of bone formation in animals with BMSCS transplanted was relatively more than those without BMSCS, and C57BL/6J mice showed more bone formation than ApoE-/-mice.Conclusions:1. BMSCs with stable morphology can be obtained by bone marrow adherent method and possess strong colony forming ability and multipotent differentiation potential. GFP transfected in BMSCs expressed stably as the cell passage increased. 2. Systemically transplanted BMSCs can home to the bone defect, differentiate into osteoblasts and promote local bone regeneration. The homing efficiency and bone regeneration capacity of systemically transplanted BMSCs were compromised in the presence of hyperlipidemia.